1 . Field of the Invention
The invention refers to a method for isolating an intestinal protein involved in intestinal cholesterol absorption and the ability to bind to cholesterol absorption inhibitors.
2 . Description of the Prior Art
In humans, on average, about 50% of the cholesterol is present in the lumen of the intestine. The intraluminal cholesterol originates mainly from the diet and from the bile. About 2 g of cholesterol a day is discharged from the bile. The intestinal cholesterol absorption depends greatly on the presence of bile salts. Thus, the effect of administration of inhibitors of the reuptake of bile salts or of bile salt sequestrants is to inhibit intestinal cholesterol absorption.
Inhibition of intestinal cholesterol absorption is an important aim of the treatment of lipid disorders, arteriosclerosis and cardiovascular disorders. The prevailing opinion amongst experts is that intestinal cholesterol absorption takes place by physicochemical diffusion.
A number of observations in connection with cholesterol transport which indicate that a protein is involved are known. Intestinal cholesterol absorption is subject to great individual variability. Biochemical data from in vitro experiments indicate that proteins are involved in cholesterol exchange between small unilamellar vesicles and the brush border vesicles of the intestine. It was possible to observe large differences in the intestinal absorption of plant sterols such as β-sitosterol and campesterol, which differ only in a methyl group (β-sitosterol) and an ethyl group (campesterol). In humans, β-sitosterol showed, inter alia, an inhibition of cholesterol absorption. There are two highly active classes of compounds which inhibit intestinal cholesterol absorption on luminal administration. The compounds are, on the one hand, compounds derived from saponin, such as tiqueside and pamaqueside, and on the other hand certain derivatives of 2-azetidinones. Derivatives of 2-azetidinones as inhibitors of cholesterol absorption are described in Clader et al., J. Med. Chem. 39, 3684-3693, 1996. For the purposes of this invention, absorption is intended to mean attachment of a substance to a protein and transport of this substance with the aid of this protein.
Intestinal absorption of cholesterol significantly contributes to serum cholesterol homeostasis. Inhibitors of intestinal cholesterol absorption like Ezetimibe or Pamaqueside have proven their efficacy as novel cholesterol-lowering agents in clinical trials. Their molecular mode of action as well as the mechanisms of intestinal cholesterol absorption are, despite enormous scientific efforts, still unknown and discussed controversially. Generally, a passive diffusion of cholesterol across plasma membranes and the intestinal brush border cell membrane is assumed, but there is increasing evidence for a protein-mediated process for intestinal cholesterol absorption. Cholesterol absorption shows a strong species difference and structurally closely related plant sterols like β-sitosterol or campesterol with comparable physico-chemical characteristics are in contrast to cholesterol only poorly absorbed making a simple diffusion process unlikely. The existence of specific transport inhibitors for cholesterol, the 2-azetidinones and sterol glycosides, with profound structure-activity-relationships strongly suggest a protein-mediated process for intestinal cholesterol absorption.
Cholesterol is a versatile compound that is vital (in small amounts) to the functioning of the human body. Only animals produce it; no plant product contains cholesterol unless an animal-based product, such as lard, has been added to it in processing. In humans, cholesterol serves three main functions. It is used by certain glands to manufacture steroid or cortisone-like hormones, including sex hormones. It helps the liver to produce bile acids, which are essential to the digestion of fats. Last but not least, it is a main component of cell membranes and structures, a kind of building block for bodily tissues. Without cholesterol, mammalian life would not exist.
The problem with cholesterol arises when the body has too much of it, or has deposits of it in the wrong places. Coronary heart disease results when cholesterol is deposited inside the walls of the heart's coronary arteries, the main suppliers of oxygen to the heart's own muscle tissue. There it contributes to the formation of fatty, toughened blockages called plaque. This buildup of plaque is variously called arteriosclerosis, hardening of the arteries, and arteriosclerosis. Cholesterol can also be deposited within arteries elsewhere in the body, where it may contribute to the occurrence of stroke (from blocked arteries in the brain) and peripheral vascular disease (from arterial blockage in the legs).
In order to travel throughout the body, cholesterol must be packaged in special molecules called lipoproteins. The lipids, or fatty cholesterol components, are wrapped inside a water-soluble protein coat. Different types of lipoproteins contain varying lipoproteins from a dynamic economy within the body, transporting cholesterol to some tissues and removing it from others. The main cholesterol-carrying compound in the body is low-density lipoprotein, or LDL-cholesterol. LDL is often referred to as the “bad cholesterol” because it appears to play a key role in depositing cholesterol within arteries. It's called low-density because it has very little protein, the most dense contents of the molecule, and is composed mainly of fats. High levels of LDL are linked to an increased risk of coronary heart disease. High-density lipoprotein, or HDL, is often termed “good cholesterol” because it appears to help remove cholesterol from artery walls and transport it to the liver for excretion. In contrast to LDL cholesterol, low levels of HDL are associated with an increased risk of coronary heart disease, while higher levels of HDL appear to protect against the disease.
Other subtypes of cholesterol particles include chylomicrons, which are produced by intestinal cells when fat is digested, and very-low-density lipoprotein (VLDL), manufactured by the liver as an important precursor of LDL cholesterol production. VLDL is the major lipoprotein that transports the triglycerides produced by the liver.
For the purpose of determining heart disease risk, LDL and HDL are key.
Given all the evidence that high-fat, high-cholesterol diets contribute to elevated levels of cholesterol in the blood, and that high blood cholesterol is a definite risk factor for heart disease, it might seem natural to assume that lowering blood cholesterol, by diet or other means, will reduce that risk.